CN1286973C - Histone methyl transferase and its preparing method - Google Patents

Abstract

The present invention provides novel histone methyltransferase-HSPC069SET protein, polynucleotide used for coding the HSPC069SET protein and a method for producing the HSPC069SET protein by a recombination technology. The present invention also discloses the usage of the polynucleotide used for coding the HSPC069SET protein. The HSPC069SET protein has the function of histone methyl transfer.

Description

A kind of ZNFN3A1 and preparation method thereof

Technical field

The invention belongs to biology field, specifically, the present invention relates to the polynucleotide of new coding human histone methyltransgerase HSPC069SET, and the polypeptide of this polynucleotide encoding.The invention still further relates to the purposes and the preparation of these polynucleotide and polypeptide.

Background technology

The posttranslational modification of histone is significant for chromosome structure and the active regulation and control of genetic transcription.These modifications mainly comprise acetylize, phosphorylation, methylate, ADP ribosylation and ubiquitinization.

Over the past two years, histone methylated functional study had obtained very big breakthrough, particularly for the methylated research of histone H 3.For example, the 9th Methionin of histone H 3 methylate with the inactivation associated of gene ( Science.2001,293:1150-5); And the 4th Methionin of histone H 3 methylate with the activation associated of gene ( Science.2001,293:2453-5).In the constitutive heterochromatin zone, the methylated histone H 3 of K9 can raise heterochromatin albumen HP1 ( Nature.2001,410:116-20): and H3-K4 methylate extensively be distributed in euchromatin ( Nat Genet.2002,30:73-6), it can stop the aminoterminal of NuRD complex body bonding histone H3, can also suppress methylating of H3-K9, thereby maintenance gene transcription activity ( Genes Dev.2002,16:479-89).

Methylating of histone all finished by ZNFN3A1.Tens histone H 3 methyltransgerases have been separated in the middle of a plurality of species from the yeast to people at present.Their common feature is all to contain the SET structural domain, and has the selectivity in histone methylated site.They can regulatory gene transcriptional activity: on the one hand, be to work by adorned histone; On the other hand, they itself are included in the protein complexes often, can make them accurately be positioned at chromosomal some section like this, and can be in the network of signal conduction functionating.For example, Rb albumen can be raised the specific ZNFN3A1 Suv39h1 of H3-K9 and the HP1 promoter region to cyclin E gene, herein the histone H 3 of methylating, thus suppress cyclin E gene transcription.If the SUV39 transgenation, then Rb albumen can not suppress cyclin E gene transcription ( Nature.2001,412:561-5).

Nearest some studies confirm that the sudden change of ZNFN3A1 can cause some defective of living body functional.The mouse chromosome instability of Suv39h disappearance, the endocellular chromosome number is irregular, tumorigenic probability raises, viability seriously reduce ( Cell.2001,107:323-37); The mice embryonic hypoevolutism of another histone H 3 methyltransgerase G9a disappearance and early stage causing death ( Genes Dev.2002,16:1779-91).This shows that all ZNFN3A1 has important regulation to the growth of organism.

In addition, ZNFN3A1 is also relevant with human diseases.The dystopy that is positioned at the mll gene of karyomit(e) 11q23 is common in the various acute leukaemic, this gene 3 ' end contains the SET structural domain, has the H3-K4 methyl transferase activity, can activate Hox a9 expression of gene, the fusion rotein of expressing after the dystopy has been lost the SET structural domain, thereby lost methyl transferase activity, this mechanism may be relevant with leukemic morbidity ( Mol Cell.2002,10:1107-17).

The ZNFN3A1 of finding is not eclipsed on function at present.At first, they can modify the different loci of histone specifically, can K4, K9, K27, K36, K79 be arranged by methylated Methionin on the known histone H 3, and wherein the methylated function of K27, K36 it be unclear that.Secondly, the methyltransgerase of the same loci that methylates is difference to some extent on Subcellular Localization often, and be in the different protein complexes, points out them to have different functions.

In sum, in view of ZNFN3A1 in vital role, therefore, this area presses for the new ZNFN3A1 of exploitation.

Summary of the invention

The purpose of this invention is to provide a kind of new human histone methyltransgerase (HSPC069SET albumen) with and fragment, analogue and derivative.

Another object of the present invention provides the polynucleotide of these polypeptide of coding.

Another object of the present invention provides the method for these polypeptide of production and the purposes of this polypeptide and encoding sequence.

In a first aspect of the present invention, novel isolated HSPC069SET polypeptide is provided, it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative of SEQID NO:2 aminoacid sequence, supplementary condition are that described polypeptide does not have the aminoacid sequence shown in the SEQ ID NO:6.

Preferably, this polypeptide is selected from down group:

(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence;

(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have the histone methyl forwarding function by (a) polypeptides derived.

More preferably, this polypeptide is the polypeptide with SEQ ID NO:2 or 4 aminoacid sequences.

In a second aspect of the present invention, the polynucleotide of isolating these polypeptide of coding are provided, these polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned people HSPC069SET polypeptide of coding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in SEQ ID NO:2 or 4.More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence that (a) has 72-3704 position among the SEQ ID NO:1; (b) has the sequence of 1-6731 position among the SEQ ID NO:1; Or (c) has a sequence of 1-1614 position among the SEQ ID NO:3.

In a third aspect of the present invention, the carrier that contains above-mentioned polynucleotide is provided, and has been transformed or host cell of transduceing or the host cell that is directly transformed or transduce by above-mentioned polynucleotide by this carrier.

In a fourth aspect of the present invention, provide preparation to have the method for the polypeptide of people HSPC069SET protein-active, this method comprises: (a) under the proteic condition of suitable expressing human HSPC069SET, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate polypeptide with people HSPC069SET protein-active.

In a fifth aspect of the present invention, provide and above-mentioned people HSPC069SET polypeptid specificity bonded antibody.

In a sixth aspect of the present invention, the compound of simulation, promotion, antagonism people HSPC069SET polypeptide active is provided, and the compound that suppresses people HSPC069SET polypeptide expression.The method of screening and/or prepare these compounds also is provided.Preferably, this compound is encoding sequence or its segmental antisense sequences of people HSPC069SET polypeptide.

In a seventh aspect of the present invention, provide and whether had the proteic method of HSPC069SET in the test sample, it comprises: sample is contacted with the proteic specific antibody of HSPC069SET, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample HSPC069SET albumen.

In a eighth aspect of the present invention, a kind of disease relevant with people HSPC069SET polypeptide unconventionality expression or method of disease susceptibility of detecting is provided, this method comprises: whether have sudden change in the nucleotide sequence of detection coding said polypeptide.

In a ninth aspect of the present invention, provide the purposes of polypeptide of the present invention and encoding sequence.Polypeptide for example of the present invention can be used to screen the agonist that promotes people HSPC069SET polypeptide active, and perhaps screening suppresses the antagonist of people HSPC069SET polypeptide active or is used to the peptide finger print identification.The proteic encoding sequence of people HSPC069SET of the present invention or its fragment can be used as primer and be used for pcr amplification reaction, perhaps are used for hybridization as probe, perhaps are used to make gene chip or microarray.

In a tenth aspect of the present invention, a kind of ZNFN3A1 composition that has is provided, it contains people HSPC069SET of the present invention or the HSPC069 polypeptide and the acceptable carrier of safe and effective amount.

Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.

Description of drawings

Following accompanying drawing is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.

Fig. 1 has shown the enzymic activity of HSPC069SET and mutant protein thereof.HSPC069 among the figure is the GST-HSPC069SET fusion rotein.

Fig. 2 has shown that HSPC069SET modifies the locus specificity of histone.

Embodiment

The inventor is through extensive and deep research, albumen HSPC069 (the SEQ ID NO:5 and 6 of a kind of Unknown Function that from the CD34+ hematopoietic stem, clones earlier, ORF is positioned at the 72-6254 position), separate first again then and obtained a kind of new ZNFN3A1 HSPC069SET (SEQ ID NO:1 and 2), this enzyme has the activity that histone methyl shifts, not only can methylate the specifically Methionin of the 36th of histone H 3 also can make himself to methylate.Finished the present invention on this basis.

In the present invention, term " HSPC069SET albumen ", " HSPC069SET polypeptide " or " ZNFN3A1 HSPC069SET " are used interchangeably, and all refer to have albumen or the polypeptide of human histone methyltransgerase HSPC069SET aminoacid sequence (SEQ ID NO:2).They comprise the ZNFN3A1 HSPC069SET that contains or do not contain initial methionine.Comprise that also to have ZNFN3A1 active and contain active fragments and fusion rotein derived from the SET structural domain of SEQ ID NO:2.

As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.

As used herein, " isolating HSPC069SET albumen or polypeptide " is meant that the HSPC069SET polypeptide is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying HSPC069SET albumen of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity of HSPC069SET polypeptide can be used amino acid sequence analysis.

Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.

The present invention also comprises the proteic fragment of people HSPC069SET, derivative and analogue.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of natural human HSPC069SET albumen of the present invention or active polypeptide basically.Polypeptide fragment of the present invention, derivative or analogue can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferred conservative amino acid residue), and the amino-acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino-acid residues, has a polypeptide of substituted radical, or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period, polyoxyethylene glycol for example) merges formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying, or with the fusion rotein of the segmental formation of antigen I gG).According to the instruction of this paper, these fragments, derivative and analogue belong to the known scope of those skilled in the art.

In the present invention, term " people HSPC069SET polypeptide " refers to have the SEQID NO:2 polypeptide of sequence of people HSPC069SET protein-active.This term also comprises having and variant form people HSPC069SET albumen identical function, SEQ IDNO:2 sequence.These variant forms comprise (but being not limited to): one or more (it is individual to be generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of people HSPC069SET and reactive derivative.

The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of people HSPC069SET DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people HSPC069SET polypeptide to obtain.The present invention also provides other polypeptide, as comprises people HSPC069SET polypeptide or its segmental fusion rotein (fusion rotein shown in SEQ ID NO:4).Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people HSPC069SET polypeptide.Usually, this fragment have people HSPC069SET peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.

Invention also provides the analogue of people HSPC069SET albumen or polypeptide.The difference of these analogues and natural human HSPC069SET polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.

In the present invention, " people HSPC069SET albumen conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO:2, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.

Table 1

Initial residue	Representational replacement	The preferred replacement
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp

Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.

The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.

Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide that also comprise additional code and/or non-coding sequence.

The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.

The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1% Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.

The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or the proteic polynucleotide of separation coding HSPC069SET.

Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.

People HSPC069SET Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.

In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.

At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.

Use method (Saiki, the et al.Science 1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.When particularly being difficult to obtain the cDNA of total length from the library, can preferably use RACE method (the terminal rapid amplifying method of RACE-cDNA), the primer that is used for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.

The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or HSPC069SET albumen coded sequence, and the method that produces polypeptide of the present invention through recombinant technology.

Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the HSPC069SET polypeptide of reorganization.In general following steps are arranged:

(1). with the polynucleotide (or varient) of coding of the present invention people HSPC069SET polypeptide, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;

(2). the host cell of in suitable medium, cultivating;

(3). separation, protein purification from substratum or cell.

Among the present invention, people HSPC069SET polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on T7 of in bacterium, expressing (Rosenberg, et al.Gene, 1987,56:125); The pMSXND expression vector of in mammalian cell, expressing (Lee and Nathans, J Bio Chem.263:3521,1988) and at the carrier that derives from baculovirus of expressed in insect cells.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.

Method well-known to those having ordinary skill in the art can be used to make up and contains people HSPC069SET DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory Manual, coldSpring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage P _LPromotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.

In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.

Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.

Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS, 293 cells or Bowes melanoma cells etc.

When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.

Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.

Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results ₂Method is handled, and used step is well-known in this area.Another kind method is to use MgCl ₂If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.

The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.

The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.

The people HSPC069SET albumen or the polypeptide of reorganization are of use in many ways.These purposes include, but is not limited to: the direct disease due to the low or forfeiture and be used to screen and promote or antibody, polypeptide or other part of antagonism HSPC069SET protein function as pharmacological agent HSPC069SET protein function.The peptide molecule that can suppress or stimulate people HSPC069SET protein function that can be used for seeking therapeutic value with the recombinant human HSPC069SET protein screening peptide library of expressing.

On the other hand, the present invention also comprises people HSPC069SET DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people HSPC069SET gene product or fragment.Preferably, refer to that those can combine with people HSPC069SET gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of people HSPC069SET, comprise that also those do not influence the antibody of people HSPC069SET protein function.The present invention also comprise those can with modify or without the people HSPC069SET gene product bonded antibody of modified forms.

The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab) ₂Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule (people such as Ladner, U.S. Patent No. 4,946,778); Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.

Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the people HSPC069SET gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human HSPC069SET albumen or its have antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology prepare (see people such as Kohler, Nature256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block people HSPC069SET protein function and the antibody that does not influence people HSPC069SET protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people HSPC069SET gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of people HSPC069SET gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).

The proteic antibody of anti-people HSPC069SET can be used in the immunohistochemistry technology, detects the people HSPC069SET albumen in the biopsy specimen.

Antibody among the present invention can be used for treating or prevention and the relevant disease of people HSPC069SET albumen.The antibody that gives suitable dosage can stimulate or block proteic generation of people HSPC069SET or activity.

Antibody also can be used for being designed to the immunotoxin at a certain privileged sites in the body.As the monoclonal antibody of people HSPC069SET albumen high-affinity can with bacterium or plant poison (as diphtheria toxin, ricin, abrine etc.) covalent attachment.A kind of usual method is with sulfydryl linking agent such as SPDP, attacks the amino of antibody, by the exchange of disulfide linkage, toxin is incorporated on the antibody, and this hybrid antibody can be used for killing the cell of people HSPC069SET protein positive.

The production of polyclonal antibody can choose HSPC069SET albumen or polypeptide immune animal, as rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.

Utilize albumen of the present invention,, can filter out with HSPC069SET albumen interactional material takes place, as acceptor, inhibitor, agonist or antagonist etc. by various conventional screening methods.

Albumen of the present invention and antibody thereof, inhibitor, agonist, antagonist or acceptor etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous or topical.

Polypeptide of the present invention can be directly used in disease treatment, for example, is used for the treatment of the low disease that causes of histone H 3 methyl transferase activity.

The present invention also provides a kind of pharmaceutical composition, and it contains HSPC069SET polypeptide of the present invention and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.

When making pharmaceutical composition, be that HSPC069SET albumen with safe and effective amount is applied to Mammals, wherein this safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.

The proteic polynucleotide of people HSPC069SET also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because cell proliferation, growth or the metabolic disturbance due to the proteic expression of HSPC069SET of the proteic nothing expression of HSPC069SET or unusual/non-activity.The HSPC069SET albumen that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic HSPC069SET protein-active.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the HSPC069SET transgenosis to cell.The method that structure carries the recombinant viral vector of HSPC069SET gene is found in existing document (Sambrook, et al.).Recombinant human HSPC069SET gene can be packaged in the liposome in addition, and then is transferred in the cell.

Suppress the oligonucleotide (comprising sense-rna and DNA) of people HSPC069SET mRNA and ribozyme also within the scope of the invention.Ribozyme is the enzyme sample RNA molecule that a kind of energy specificity is decomposed specific RNA, and its mechanism of action is to carry out the endonuclease effect after ribozyme molecule and the hybridization of complementary target RNA-specific.The RNA of antisense and DNA and ribozyme can obtain with existing any RNA or DNA synthetic technology, as the technology widespread use of solid phase phosphoamide chemical synthesis synthetic oligonucleotide.Antisense rna molecule can be transcribed acquisition by the dna sequence dna of this RNA that encodes in external or body.This dna sequence dna has been incorporated into the downstream of rna polymerase promoter of carrier.In order to increase the stability of nucleic acid molecule, available several different methods is modified it, and as increasing the sequence length of both sides, the connection between the ribonucleoside is used phosphoric acid thioester bond or peptide bond but not phosphodiester bond.

Polynucleotide imports tissue or intracellular method comprises: directly be injected into polynucleotide in the in-vivo tissue; Or external by carrier (as virus, phage or plasmid etc.) earlier with the polynucleotide transfered cell in, again cell is transplanted in the body etc.

Can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with the protein bound peptide molecule of people HSPC069SET obtains.During screening, must carry out mark to people HSPC069SET protein molecular.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization people HSPC069SET protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The people HSPC069SET protein level that is detected in the test can be with laying down a definition the importance of people HSPC069SET albumen in various diseases and be used to the disease of diagnosing HSPC069SET albumen to work.

Whether having the proteic method of HSPC069SET in a kind of detection test sample is to utilize the proteic specific antibody of HSPC069SET to detect, and it comprises: sample is contacted with the HSPC069SET protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample HSPC069SET albumen.

The proteic polynucleotide of HSPC069SET can be used for the diagnosis and the treatment of HSPC069SET protein related diseases.Aspect diagnosis, the proteic polynucleotide of HSPC069SET can be used for detecting the proteic expression of HSPC069SET HSPC069SET abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of HSPC069SET as the HSPC069SET dna sequence dna.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of HSPC069SET albumen and also can detect the proteic transcription product of HSPC069SET.

The sudden change that detects the HSPC069SET gene also can be used for the disease of diagnosing HSPC069SET albumen relevant.The form of HSPC069SET protein mutation comprises that the point mutation compared with normal wild type HSPC069SET dna sequence dna, transposition, disappearance, reorganization and other are any unusual etc.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.

Sequence of the present invention identifies it also is valuable to karyomit(e).In brief, the proteic cDNA of HSPC069SET prepares PCR primer (preferred 15-35bp) according to the present invention, sequence can be positioned on the karyomit(e).Then, these primers are used for the somatocyte hybrid cell that the PCR screening contains each bar human chromosome.Have only those hybrid cells that contain corresponding to the people's gene of primer can produce the fragment of amplification.

In case sequence is positioned to chromosome position accurately, the physical location of this sequence on karyomit(e) just can be associated with the gene map data.These data for example are found in, V.Mckusick, Mendelian Inheritance inMan (can by with the online acquisition of Johns Hopkins University Welch Medical Library).Can pass through linkage analysis then, determine gene and navigated to relation between the disease on the chromosomal region already.

In an example of the present invention, a kind of isolating polynucleotide are provided, its coding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.Polynucleotide of the present invention are isolated from human hematopoietic stem cell cDNA library.Its sequence is shown in SEQ ID NO:1, and the polynucleotide sequence total length that it comprises is 6731 bases, and its open reading frame is positioned at the 72-3704 position, and the coding total length is 1211 amino acid whose people HSPC069SET albumen (SEQ ID NO:2).HSPC069SET albumen has the activity of ZNFN3A1, its can methylate specifically Methionin of the 36th of histone H 3.Before the present invention, the enzyme in this site of in human body, finding as yet to methylate.In addition, this enzyme can also methylate self, and all at present ZNFN3A1 do not possess this character.In view of the significance of ZNFN3A1 in organism developmental regulation and human diseases generation, the HSPC069SET ZNFN3A1 may be applied to treating human diseases.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

The clone of embodiment 1:HSPC069SET albumen cDNA

The cDNA fragment of HSPC069SET come from the CD34 male hematopoietic stem cDNA library that makes up with ordinary method (method is referring to Zhang et al., Genome Res.2000,10:1546-6).With the cDNA library is template, with a pair of oligonucleotide is primer-upstream: ctcagatctaacagggacctaaggacatcatc (SEQ IDNO:7) and downstream: cgcggtaccttattttcaatatattcacatatacatta (SEQ ID NO:8), carries out PCR.The fragment that amplifies is cut with the BglII/KpnI enzyme, is connected into pEGFP carrier (Clontech company), obtains the HPC069SET-pEGFP plasmid, and the nucleotide sequence of the HSPC069SET that obtains is identified in order-checking.

HSPC069SET cDNA total length is 6731bp (SEQ ID NO:1), contains complete open frame (72-3704 position), and coding contains the polypeptide (SEQ ID NO:2) of 1211 amino-acid residues.Homology relatively shows and contains in the evolution as follows conservative SET structural domain in this section aminoacid sequence:

FQRKQHADVE VILTEKKGWG LRAAKDLPSN TFVLEYCGEV LDHKEFKARV 50

KEYARNKNIH YYFMALKNDE IIDATQKGNC SRFMNHSCEP NCETQKWTVN 100

GQLRVGFFTT KLVPSGSELT FDYQFQRYGK 130

(being 1041-1170 position among the SEQ ID NO:2).

Embodiment 2

Expression and the purifying of HSPC069SET albumen in intestinal bacteria

With extractive hemopoietic stem cell mRNA is template, after reverse transcription, with a pair of oligonucleotide is primer-A:gcgtcgacgtgatggtgagcttcaggacaga (SEQ ID NO:9) and B:aactgcagatgtgaggcagacaagtcattcca (SEQ ID NO:10), carries out PCR.The fragment that amplifies is cut with restriction endonuclease SalI/PstI enzyme in restricted, and product is connected into pGBKT7 (available from Clontech company), obtains the HPC069SET-pGBKT7 plasmid, and the cDNA that obtains is identified in order-checking.

Then, after HPC069SET-pGBKT7 cut with restriction enzyme EcoRI enzyme, be connected into pGEX-5X1 carrier (Amersham Biosciences), obtain the HSPC069SET-pGEX-5X1 plasmid.The cDNA fragment of order-checking evaluation HSPC069SET is correctly inserted carrier.With this plasmid transformation escherichia coli BL21 bacterial strain.Positive transformant is overnight incubation in containing the LB substratum of Amp, is transferred in the substratum of large volume with 1: 100 ratio then, is cultured to 600 nanometer optical density(OD) and reaches 0.6-1.0, adds IPTG to final concentration 0.5mM, 28 degrees centigrade of abduction deliverings 3 hours.Collecting cell is also used the ultrasonic degradation cell.Add glutathione S epharose 4B pearl in the supernatant liquor, rotation mixing 1 hour, centrifugal collection pearl is also washed 3 times with the PBS damping fluid, and it is standby at last the albumen on the pearl to be kept at-80 degree.SDS-PAGE gel electrophoresis with 12% is identified and is obtained proteic molecular weight.

Measurement result shows that the molecular weight of fusion rotein is 62Kda, conforms to expection.(expressed proteins is the GST-HSPC069SET fusion rotein, and its encoding sequence and aminoacid sequence are shown in SEQ ID NO:3 and 4.)

Embodiment 3

The structure of mutant and the acquisition of mutant protein

With the method for the bridge-type PCR of routine, on the basis of the HSPC069SET-pGEX-5X1 plasmid of embodiment 2 preparations, the 1122nd arginine in the proteic SET structural domain of HSPC069SET sported Histidine (be among the SEQ ID NO:4 the 449th).By with embodiment 2 same procedure, obtain the HSPC069SETMp albumen of sudden change with this mutant plasmid,

Embodiment 4

The detection of HSPC069SET protease activity

Adopting the GST-fusion rotein to detect enzymic activity is present method in common.With the GST-fusion rotein GST-mG9a (amino acid 621-1000) of known ZNFN3A1, GST-SUV39H1 (amino acid 82-412) and GST-MLL (amino acid 3745-3699) use the histone mixture as substrate as positive control.GST-HSPC069SET albumen or positive control albumen are mixed with substrate, methyl donor S-adenosine-[methyl isophthalic acid the 4C]-L-methionine(Met) (PerkinElmer Life Sciences company) and reaction buffer (the 50mM Tris pH8.5 that add carbon 14 marks, 20mM KCl, 10mM MgCl2,1% beta-mercaptoethanol, 250mM sucrose), hatched 1 hour for 37 ℃, reaction product NuPAGE Novex Bis-Tris gel (Invitrogen company) electrophoresis, with behind the coomassie brilliant blue staining gel being drained, detect the isotropic substance autography with Molecular Imager FX (Bio-Rad company).

The result shows that GST-HSPC069SET albumen has tangible enzymic activity, can modify histone H 3 (Figure 1A.Annotate: the HSPC069=GST-HSPC069SET fusion rotein).If substitute GST-HSPC069SET albumen with mutant GST-HSPC069SETMp albumen, then enzymic activity disappears (Fig. 1 C), illustrates that this activity depends on the SET structural domain.

In order to determine the locus specificity of the protein modified histone H 3 of HSPC069SET, adopt the various mutant GST-H3N of the histone H 3 of reorganization, N4, N9, N27 and K36R be as substrate (Fig. 2 A), and the result shows that GST-HSPC069SET albumen can modify the 36th Methionin (Fig. 2 B) of histone H 3 specifically.

Except modifying the histone, HSPC069SET albumen can also methylate self.No matter whether the substrate histone exist, GST-HSPC069SET all can be methylated, and illustrates that this self methylated activity of HSPC069SET does not rely on exist (Figure 1A, B, C, asterisk represent self methylated signal) of histone.All ZNFN3A1 of having reported at present all do not find to have this kind activity.

The generation of embodiment 5 anti-HSPC069SET protein antibodies

The recombinant human HSPC069SET albumen that obtains among the embodiment 2 is used for immune animal to produce antibody, and concrete grammar is as follows.Recombinant molecule is standby after separating with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days,, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation people HSPC069SET protein gene translation product with it.Found that antibody can combine with albumen of the present invention specifically.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table

＜110〉Ruijin Hospital Attached to Shanghai Medical Univ No.2

＜120〉a kind of ZNFN3A1 and preparation method thereof

<130>041666

<160>10

<170>PatentIn version 3.1

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＜213〉homo sapiens (Homo sapiens)

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<221>CDS

<222>(72)..(3704)

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acagggacct aaggacatca tcctattcta aatctgatcg ggactgtaaa actgagacct 60

cttacttaga g atg gaa aga aga ggc aag tat tct tca aaa cta gaa aga 110

Met Glu Arg Arg Gly Lys Tyr Ser Ser Lys Leu Glu Arg

1 5 10

gaa tct aaa agg act tca gaa aat gaa gca att aaa aga tgt tgt tct 158

Glu Ser Lys Arg Thr Ser Glu Asn Glu Ala Ile Lys Arg Cys Cys Ser

15 20 25

ccc cct aat gaa ctg gga ttc cga cga ggg tca tca tat tct aag cat 206

Pro Pro Asn Glu Leu Gly Phe Arg Arg Gly Ser Ser Tyr Ser Lys His

30 35 40 45

gac agt agt gct tcc cgt tat aaa tct acc ctt tca aaa cct ata ccc 254

Asp Ser Ser Ala Ser Arg Tyr Lys Ser Thr Leu Ser Lys Pro Ile Pro

50 55 60

aag tct gat aaa ttt aaa aat tct ttc tgt tgt aca gaa tta aat gaa 302

Lys Ser Asp Lys Phe Lys Asn Ser Phe Cys Cys Thr Glu Leu Asn Glu

65 70 75

gaa atc aaa caa tct cat tct ttt agt tta cag aca cct tgt tca aaa 350

Glu Ile Lys Gln Ser His Ser Phe Ser Leu Gln Thr Pro Cys Ser Lys

80 85 90

ggt agt gaa tta aga atg att aat aaa aat cct gaa aga gaa aag gct 398

Gly Ser Glu Leu Arg Met Ile Asn Lys Asn Pro Glu Arg Glu Lys Ala

95 100 105

ggg tct cca gct cca tca aat cga tta aat gat tca cct act tta aaa 446

Gly Ser Pro Ala Pro Ser Asn Arg Leu Asn Asp Ser Pro Thr Leu Lys

110 115 120 125

aag cta gat gaa ttg cct att ttt aag tcc gaa ttt ata aca cat gat 494

Lys Leu Asp Glu Leu Pro Ile Phe Lys Ser Glu Phe Ile Thr His Asp

130 135 140

agc cat gat agt att aag gaa tta gac tct tta tct aaa gtg aag aat 542

Ser His Asp Ser Ile Lys Glu Leu Asp Ser Leu Ser Lys Val Lys Asn

145 150 155

gat caa tta aga agt ttt tgt ccc ata gaa tta aat ata aat gga tct 590

Asp Gln Leu Arg Ser Phe Cys Pro Ile Glu Leu Asn Ile Asn Gly Ser

160 165 170

cct ggg gca gaa tct gat ttg gca aca ttt tgc act tct aaa act gat 638

Pro Gly Ala Glu Ser Asp Leu Ala Thr Phe Cys Thr Ser Lys Thr Asp

175 180 185

gct gtt tta atg act tct gat gat agt gtg act gga tcg gaa tta tcc 686

Ala Val Leu Met Thr Ser Asp Asp Ser Val Thr Gly Ser Glu Leu Ser

190 195 200 205

cct ttg gtc aaa gca tgc atg ctt tca tca aat gga ttt cag aat att 734

Pro Leu Val Lys Ala Cys Met Leu Ser Ser Asn Gly Phe Gln Asn Ile

210 215 220

agt agg tgc aaa gaa aaa gac ttg gat gat acc tgc atg ctg cat aag 782

Ser Arg Cys Lys Glu Lys Asp Leu Asp Asp Thr Cys Met Leu His Lys

225 230 235

aag tca gaa agc cca ttt aga gaa aca gaa cct ctg gtg tca cca cac 830

Lys Ser Glu Ser Pro Phe Arg Glu Thr Glu Pro Leu Val Ser Pro His

240 245 250

caa gat aaa ctc atg tct atg cca gtt atg act gtg gat tat tcc aaa 878

Gln Asp Lys Leu Met Ser Met Pro Val Met Thr Val Asp Tyr Ser Lys

255 260 265

aca gta gtt aaa gaa cca gtt gat acg agg gtt tct tgc tgc aaa acc 926

Thr Val Val Lys Glu Pro Val Asp Thr Arg Val Ser Cys Cys Lys Thr

270 275 280 285

aaa gat tca gac ata tac tgt act ttg aac gat agc aac cct tct ttg 974

Lys Asp Ser Asp Ile Tyr Cys Thr Leu Asn Asp Ser Asn Pro Ser Leu

290 295 300

tgt aac tct gaa gct gaa aat att gag cct tca gtt atg aag att tct 1022

Cys Asn Ser Glu Ala Glu Asn Ile Glu Pro Ser Val Met Lys Ile Ser

305 310 315

tca aat agc ttt atg aat gtg cat ttg gaa tca aaa cca gtt ata tgt 1070

Ser Asn Ser Phe Met Asn Val His Leu Glu Ser Lys Pro Val Ile Cys

320 325 330

gat agt aga aat ttg aca gat cac tca aaa ttt gca tgt gaa gaa tat 1118

Asp Ser Arg Asn Leu Thr Asp His Ser Lys Phe Ala Cys Glu Glu Tyr

335 340 345

aag cag agc atc ggt agc act agt tca gct tct gtt aat cat ttt gat 1166

Lys Gln Ser Ile Gly Ser Thr Ser Ser Ala Ser Val Asn His Phe Asp

350 355 360 365

gat tta tat caa cct att ggg agt tca ggt att gct tca tct ctt cag 1214

Asp Leu Tyr Gln Pro Ile Gly Ser Ser Gly Ile Ala Ser Ser Leu Gln

370 375 380

agt ctt cca cca gga ata aag gtg gac agt cta act ctc ttg aaa tgc 1262

Ser Leu Pro Pro Gly Ile Lys Val Asp Ser Leu Thr Leu Leu Lys Cys

385 390 395

gga gag aac aca tct cca gtt ctg gat gca gtg cta aag agt aaa aaa 1310

Gly Glu Asn Thr Ser Pro Val Leu Asp Ala Val Leu Lys Ser Lys Lys

400 405 410

agt tca gag ttt tta aag cat gca ggg aaa gaa aca ata gta gaa gta 1358

Ser Ser Glu Phe Leu Lys His Ala Gly Lys Glu Thr Ile Val Glu Val

415 420 425

ggt agt gac ctt cct gat tca gga aag gga ttt gct tcc agg gag aac 1406

Gly Ser Asp Leu Pro Asp Ser Gly Lys Gly Phe Ala Ser Arg Glu Asn

430 435 440 445

agg cgt aat aat ggg tta tct ggg aaa tgt ttg caa gag gct caa aaa 1454

Arg Arg Asn Asn Gly Leu Ser Gly Lys Cys Leu Gln Glu Ala Gln Lys

450 455 460

gaa ggg aat tcc ata ttg cct gaa aga aga gga aga cca gaa atc tct 1502

Glu Gly Asn Ser Ile Leu Pro Glu Arg Arg Gly Arg Pro Glu Ile Ser

465 470 475

tta gat gaa aga gga gaa gga gga cat gtg cat act tct gat gac tca 1550

Leu Asp Glu Arg Gly Glu Gly Gly His Val His Thr Ser Asp Asp Ser

480 485 490

gaa gtt gta ttt tct tct tgt gat ttg aat tta acc atg gaa gac agt 1598

Glu Val Val Phe Ser Ser Cys Asp Leu Asn Leu Thr Met Glu Asp Ser

495 500 505

gat ggt gta act tat gca tta aag tgt gac agt agt ggt cat gcc cca 1646

Asp Gly Val Thr Tyr Ala Leu Lys Cys Asp Ser Ser Gly His Ala Pro

510 515 520 525

gaa att gtg tct aca gtt cat gaa gat tat tct ggc tct tct gaa agt 1694

Glu Ile Val Ser Thr Val His Glu Asp Tyr Ser Gly Ser Ser Glu Ser

530 535 540

tca aat gat gaa agt gat tca gaa gat acg gat tcg gat gat agc agt 1742

Ser Asn Asp Glu Ser Asp Ser Glu Asp Thr Asp Ser Asp Asp Ser Ser

545 550 555

att cca asa aac cgt ctc cag tct gtt gtg gtt gtg cca aag aat tct 1790

Ile Pro Arg Asn Arg Leu Gln Ser Val Val Val Val Pro Lys Asn Ser

560 565 570

act ttg ccc atg gaa gaa aca agt cct tgt tct tct cgg agc agt caa 1838

Thr Leu Pro Met Glu Glu Thr Ser Pro Cys Ser Ser Arg Ser Ser Gln

575 580 585

agt tat aga cac tat tct gac cat tgg gaa gat gag aga ttg gag tca 1886

Ser Tyr Arg His Tyr Ser Asp His Trp Glu Asp Glu Arg Leu Glu Ser

590 595 600 605

agg aga cat ttg tat gag gaa aaa ttt gaa agt ata gca agt aaa gcc 1934

Arg Arg His Leu Tyr Glu Glu Lys Phe Glu Ser Ile Ala Ser Lys Ala

610 615 620

tgt cct caa act gat aag ttt ttc ctt cat aaa gga aca gag aag aat 1982

Cys Pro Gln Thr Asp Lys Phe Phe Leu His Lys Gly Thr Glu Lys Asn

625 630 635

ccg gaa att tct ttt aca cag tcc agt aga aaa caa ata gat aac cgc 2030

Pro Glu Ile Ser Phe Thr Gln Ser Ser Arg Lys Gln Ile Asp Asn Arg

640 645 650

ctg cct gaa ctt tct cat cct cag agt gat ggg gtt gat agt aca agt 2078

Leu Pro Glu Leu Ser His Pro Gln Ser Asp Gly Val Asp Ser Thr Ser

655 660 665

cat aca gat gtg aaa tct gac cct ctg ggt cac cca aat tca gag gaa 2126

His Thr Asp Val Lys Ser Asp Pro Leu Gly His Pro Asn Ser Glu Glu

670 675 680 685

acc gtg aaa gcc aaa ata cct tct agg cag caa gaa gag ctg cca att 2174

Thr Val Lys Ala Lys Ile Pro Ser Arg Gln Gln Glu Glu Leu Pro Ile

690 695 700

tat tct tct gat ttt gaa gat gtc cca aat aag tct tgg caa cag acc 2222

Tyr Ser Ser Asp Phe Glu Asp Val Pro Asn Lys Ser Trp Gln Gln Thr

705 710 715

act ttc caa aac agg cca gat agt aga ctg gga aaa aca gaa ttg agt 2270

Thr Phe Gln Asn Arg Pro Asp Ser Arg Leu Gly Lys Thr Glu Leu Ser

720 725 730

ttt tct tcc tct tgt gag ata cca cat gtg gat ggc ttg cac tca tca 2318

Phe Ser Ser Ser Cys Glu Ile Pro His Val Asp Gly Leu His Ser Ser

735 740 745

gaa gag ctc aga aac tta ggt tgg gac ttc tct caa gaa aag cct tct 2366

Glu Glu Leu Arg Asn Leu Gly Trp Asp Phe Ser Gln Glu Lys Pro Ser

750 755 760 765

gcc acg tat cag caa cct gac agt agc tat gga gct tgt ggt gga cac 2414

Ala Thr Tyr Gln Gln Pro Asp Ser Ser Tyr Gly Ala Cys Gly Gly His

770 775 780

aag tat cag caa aat gca gaa cag tat ggt ggg aca cgt gat tac tgg 2462

Lys Tyr Gln Gln Asn Ala Glu Gln Tyr Gly Gly Thr Arg Asp Tyr Trp

785 790 795

caa ggc aat ggt tac tgg gat cca aga tca ggt aga cct cct gga act 2510

Gln Gly Asn Gly Tyr Trp Asp Pro Arg Ser Gly Arg Pro Pro Gly Thr

800 805 810

ggg gtt gtg tat gat cga act caa gga caa gta cca gat tcc cta aca 2558

Gly Val Val Tyr Asp Arg Thr Gln Gly Gln Val Pro Asp Ser Leu Thr

815 820 825

gat gat cgt gaa gaa gag gag aat tgg gat caa cag gat gga tcc cat 2606

Asp Asp Arg Glu Glu Glu Glu Asn Trp Asp Gln Gln Asp Gly Ser His

830 835 840 845

ttt tca gac cag tcc gat aaa ttt ctt cta tcc ctt cag aaa gac aag 2654

Phe Ser Asp Gln Ser Asp Lys Phe Leu Leu Ser Leu Gln Lys Asp Lys

850 855 860

ggg tca gtg caa gca cct gaa ata agc agc aat tcc att aag gac act 2702

Gly Ser Val Gln Ala Pro Glu Ile Ser Ser Asn Ser Ile Lys Asp Thr

865 870 875

tta gct gtg aat gaa aag aaa gat ttt tca aaa aac tta gaa aaa aat 2750

Leu Ala Val Asn Glu Lys Lys Asp Phe Ser Lys Asn Leu Glu Lys Asn

880 885 890

gat atc aaa gat aga ggg cct ctt aaa aaa agg agg cag gaa ata gag 2798

Asp Ile Lys Asp Arg Gly Pro Leu Lys Lys Arg Arg Gln Glu Ile Glu

895 900 905

agt gat tct gaa agt gat ggt gag ctt cag gac aga aag aaa gtt aga 2846

Ser Asp Ser Glu Ser Asp Gly Glu Leu Gln Asp Arg Lys Lys Val Arg

910 915 920 925

gtg gag gta gag cag gga gag aca tca gtg ccc cca ggt tca gca ctg 2894

Val Glu Val Glu Gln Gly Glu Thr Ser Val Pro Pro Gly Ser Ala Leu

930 935 940

gtt ggg ccc tcc tgt gtc atg gat gac ttc agg gac cca cag cga tgg 2942

Val Gly Pro Ser Cys Val Met Asp Asp Phe Arg Asp Pro Gln Arg Trp

945 950 955

aag gaa tgt gcc aag caa ggg aaa atg cca tgt tac ttt gat ctt att 2990

Lys Glu Cys Ala Lys Gln Gly Lys Met Pro Cys Tyr Phe Asp Leu Ile

960 965 970

gaa gaa aat gtt tat tta aca gaa aga aag aag aat aaa tct cat cga 3038

Glu Glu Asn Val Tyr Leu Thr Glu Arg Lys Lys Asn Lys Ser His Arg

975 980 985

gat att aag cga atg cag tgt gag tgt aca cct ctt tct aaa gat gaa 3086

Asp Ile Lys Arg Met Gln Cys Glu Cys Thr Pro Leu Ser Lys Asp Glu

990 995 1000 1005

aga gct caa ggt gaa ata gca tgt ggg gaa gat tgt ctt aat cgt 3131

Arg Ala Gln Gly Glu Ile Ala Cys Gly Glu Asp Cys Leu Asn Arg

1010 1015 1020

ctt ctc atg att gaa tgt tct tct cgg tgt cca aat ggg gat tat 3176

Leu Leu Met Ile Glu Cys Ser Ser Arg Cys Pro Asn Gly Asp Tyr

1025 1030 1035

tgt tcc aat aga cgg ttt cag aga aaa cag cat gca gat gtg gaa 3221

Cys Ser Asn Arg Arg Phe Gln Arg Lys Gln His Ala Asp Val Glu

1040 1045 1050

gtc ata ctc aca gaa aag aaa ggc tgg ggc ttg aga gct gcc aaa 3266

Val Ile Leu Thr Glu Lys Lys Gly Trp Gly Leu Arg Ala Ala Lys

1055 1060 1065

gac ctt cct tcg aac acc ttt gtc cta gaa tat tgt gga gag gta 3311

Asp Leu Pro Ser Asn Thr Phe Val Leu Glu Tyr Cys Gly Glu Val

1070 1075 1080

ctc gat cat aaa gag ttt aaa gct cga gtg aag gag tat gca cga 3356

Leu Asp His Lys Glu Phe Lys Ala Arg Val Lys Glu Tyr Ala Arg

1085 1090 1095

aac aaa aac atc cat tac tat ttc atg gcc ctg aag aat gat gag 3401

Asn Lys Asn Ile His Tyr Tyr Phe Met Ala Leu Lys Asn Asp Glu

1100 1105 1110

ata ata gat gcc act caa aaa gga aat tgc tct cgt ttc atg aat 3446

Ile Ile Asp Ala Thr Gln Lys Gly Asn Cys Ser Arg Phe Met Asn

1115 1120 1125

cac agc tgt gaa cca aat tgt gaa acc caa aaa tgg act gtg aac 3491

His Ser Cys Glu Pro Asn Cys Glu Thr Gln Lys Trp Thr Val Asn

1130 1135 1140

gga caa ctg agg gtt ggg ttt ttt acc acc aaa ctg gtt cct tca 3536

Gly Gln Leu Arg Val Gly Phe Phe Thr Thr Lys Leu Val Pro Ser

1145 1150 1155

ggc tca gag tta acg ttt gac tat cag ttc cag aga tat gga aaa 3581

Gly Ser Glu Leu Thr Phe Asp Tyr Gln Phe Gln Arg Tyr Gly Lys

1160 1165 1170

gaa gcc cag aaa tgt ttc tgc gga tca gcc aat tgc cgg ggt tac 3626

Glu Ala Gln Lys Cys Phe Cys Gly Ser Ala Asn Cys Arg Gly Tyr

1175 1180 1185

ctg gga gga gaa aac aga gtc agc att aga gca gca gga ggg aaa 3671

Leu Gly Gly Glu Asn Arg Val Ser Ile Arg Ala Ala Gly Gly Lys

1190 1195 1200

atg aag aag gaa cga tct cgt aag aag gat tca taggtggatg 3714

Met Lys Lys Glu Arg Ser Arg Lys Lys Asp Ser

1205 1210

gagagctaga agctctgatg gaaaatggtg agggtctctc tgataaaaac caggtgccca 3774

gcttatcccg gctaatggtt agaattgaaa ctttggagca gaaacttacc tgtctggaac 3834

tcatacagaa cacacactca cagtcctgcc tgaagtcctt tctggaacgt catgggctgt 3894

ctttgttgtg gatctggatg gcagagctag gtgacggccg ggaaagtaac cagaagcttc 3954

aggaagagat tataaagact ttggaacact tgcccattcc tactaaaaat atgttggagg 4014

aaagcaaagt acttccaatt attcaacgct ggtctcagac taagactgct gtccctccgt 4074

tgagtgaagg agatgggtat tctagtgaga atacatcgcg tgctcataca ccactcaaca 4134

cacctgatcc ttccaccaag ctgagcacag aagctgacac agacactccc aagaaactaa 4194

tgtttcgcag actgaaaatt ataagtgaaa atagcatgga cagtgcaatc tctgatgcaa 4254

ccagtgagct agaaggcaag gatggcaaag aggatcttga tcaattagaa aatgtccctg 4314

tagaggaaga ggaagaattg cagtcacaac agctactccc acaacagctg cctgaatgca 4374

aagttgatag tgaaaccaac atagaagcta gtaagctacc tacatctgaa ccagaagctg 4434

acgctgaaat agagcccaaa gagagcaacg gcacaaaact agaagaacct attaatgaag 4494

aaacaccatc ccaagatgaa gaggagggtg tgtctgatgt ggagagtgaa aggagccaag 4554

aacagccaga taaaacagtg gatataagtg atttggccac caaactcctg gacagttgga 4614

aagacctaaa ggaggtatat cgaattccaa agaaaagtca aactgaaaag gaaaacacaa 4674

gaaatcagcc tgaataaatg gaatgacttg tctgcctcac atattctaag gtgcagagtc 4734

agaatatgaa ctgttgcaac tgaacgagga agggatgctg ttggcttcag agatcaaaca 4794

cctgccccga agactcctaa taggtcaaga gagagagacc cagacaagca aactcaaaat 4854

aaagagaaaa ggaaacgaag aagctccctc tcaccaccct cttctgccta tgagcgggga 4914

acaaaaaggc cagatgacag atatgataca ccaacttcta aaaagaaagt acgaattaaa 4974

gaccgcaata aactttctac agaggaacgc cggaagttgt ttgagcaaga ggtggctcaa 5034

cgggaggctc agaaacaaca gcaacagatg cagaacctgg gaatgacatc accactgccc 5094

tatgactctc ttggttataa tgccccgcat catccctttg ctggttaccc accaggttat 5154

cccatgcagg cctatgtgga tcccagcaac cctaatgctg gaaaggtgct cctgcccaca 5214

cccagcatgg acccagtgtg ttctcctgct ccttatgatc atgctcagcc cttggtggga 5274

cattctacag aacccctttc tgcccctcca ccagtaccag tggtgccaca tgtggcagct 5334

cctgtggaag tttccagttc ccagtatgtg gcccagagtg atggtgtagt acaccaagac 5394

tccagcgttg ctgtcttgcc agtgccggcc cccggcccag ttcagggaca gaattatagt 5454

gtttgggatt caaaccaaca gtctgtcagt gtacagcagc agtactctcc tgcacagtct 5514

caagcaacca tatattatca aggacagaca tgtccaacag tctatggtgt gacatcacct 5574

tattcacaga caactccacc aattgtacag agttatgccc agccaagtct tcagtatatc 5634

caggggcaac agattttcac agctcatcca caaggagtgg tggtacagcc agccgcagca 5694

gtgactacaa tagttgcacc agggcagcct cagcccttgc agccatctga aatggttgtg 5754

acaaataatc tcttggatct gccgcccccc tctcctccca aaccaaaaac cattgtctta 5814

cctcccaact ggaagacagc tcgagatcca gaagggaaga tttattacta ccatgtgatc 5874

acaaggcaga ctcagtggga tcctcctact tgggaaagcc caggagatga tgccagcctt 5934

gagcatgaag ctgagatgga cctgggaact ccaacatatg atgaaaaccc catgaaggcc 5994

tcgaaaaagc ccaagacagc agaagcagac acctccagtg aactagcaaa gaaaagcaaa 6054

gaagtattca gaaaagagat gtcccagttc atcgtccagt gcctgaaccc ttaccggaaa 6114

cctgactgca aagtgggaag aattaccaca actgaagact ttaaacatct ggctcgcaag 6174

ctgactcacg gtgttatgaa taaggagctg aagtactgta agaatcctga ggacctggag 6234

tgcaatgaga atgtgaaaca caaaaccaag gagtacatta agaagtacat gcagaagttt 6294

ggggctgttt acaaacccaa agaggacact gaattagagt gactgttggg ccagggtggg 6354

aggatgggtg gtcaggtaag acagactcta gggagaggaa atcctgtggg cctttctgtc 6414

ccacccctgt cagcactgtg ctactgatga tacatcaccc tggggaattc aaccctgcag 6474

atgtcaactg aaggccacaa aaatgaactc catctacaag tgattaccta gttgtgagct 6534

gttggcatgt ggttagaagc catcagaggt gcaagggctt agaaaagacc ctggccagac 6594

ctgactccac tcttaaacct gggtcttctc cttggcggtg ctgtcagcgc acagacccat 6654

gcgcatcccc acccacaacc ctttaccctg atgatctgta ttatatttta atgtatatgt 6714

gaatatattg aaaataa 6731

<210>2

<211>1211

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>2

Met Glu Arg Arg Gly Lys Tyr Ser Ser Lys Leu Glu Arg Glu Ser Lys

1 5 10 15

Arg Thr Ser Glu Asn Glu Ala Ile Lys Arg Cys Cys Ser Pro Pro Asn

20 25 30

Glu Leu Gly Phe Arg Arg Gly Ser Ser Tyr Ser Lys His Asp Ser Ser

35 40 45

Ala Ser Arg Tyr Lys Ser Thr Leu Ser Lys Pro Ile Pro Lys Ser Asp

50 55 60

Lys Phe Lys Asn Ser Phe Cys Cys Thr Glu Leu Asn Glu Glu Ile Lys

65 70 75 80

Gln Ser His Ser Phe Ser Leu Gln Thr Pro Cys Ser Lys Gly Ser Glu

85 90 95

Leu Arg Met Ile Asn Lys Asn Pro Glu Arg Glu Lys Ala Gly Ser Pro

100 105 110

Ala Pro Ser Asn Arg Leu Asn Asp Ser Pro Thr Leu Lys Lys Leu Asp

115 120 125

Glu Leu Pro Ile Phe Lys Ser Glu Phe Ile Thr His Asp Ser His Asp

130 135 140

Ser Ile Lys Glu Leu Asp Ser Leu Ser Lys Val Lys Asn Asp Gln Leu

145 150 155 160

Arg Ser Phe Cys Pro Ile Glu Leu Asn Ile Asn Gly Ser Pro Gly Ala

165 170 175

Glu Ser Asp Leu Ala Thr Phe Cys Thr Ser Lys Thr Asp Ala Val Leu

180 185 190

Met Thr Ser Asp Asp Ser Val Thr Gly Ser Glu Leu Ser Pro Leu Val

195 200 205

Lys Ala Cys Met Leu Ser Ser Asn Gly Phe Gln Asn Ile Ser Arg Cys

210 215 220

Lys Glu Lys Asp Leu Asp Asp Thr Cys Met Leu His Lys Lys Ser Glu

225 230 235 240

Ser Pro Phe Arg Glu Thr Glu Pro Leu Val Ser Pro His Gln Asp Lys

245 250 255

Leu Met Ser Met Pro Val Met Thr Val Asp Tyr Ser Lys Thr Val Val

260 265 270

Lys Glu Pro Val Asp Thr Arg Val Ser Cys Cys Lys Thr Lys Asp Ser

275 280 285

Asp Ile Tyr Cys Thr Leu Asn Asp Ser Asn Pro Ser Leu Cys Asn Ser

290 295 300

Glu Ala Glu Asn Ile Glu Pro Ser Val Met Lys Ile Ser Ser Asn Ser

305 310 315 320

Phe Met Asn Val His Leu Glu Ser Lys Pro Val Ile Cys Asp Ser Arg

325 330 335

Asn Leu Thr Asp His Ser Lys Phe Ala Cys Glu Glu Tyr Lys Gln Ser

340 345 350

Ile Gly Ser Thr Ser Ser Ala Ser Val Asn His Phe Asp Asp Leu Tyr

355 360 365

Gln Pro Ile Gly Ser Ser Gly Ile Ala Ser Ser Leu Gln Ser Leu Pro

370 375 380

Pro Gly Ile Lys Val Asp Ser Leu Thr Leu Leu Lys Cys Gly Glu Asn

385 390 395 400

Thr Ser Pro Val Leu Asp Ala Val Leu Lys Ser Lys Lys Ser Ser Glu

405 410 415

Phe Leu Lys His Ala Gly Lys Glu Thr Ile Val Glu Val Gly Ser Asp

420 425 430

Leu Pro Asp Ser Gly Lys Gly Phe Ala Ser Arg Glu Asn Arg Arg Asn

435 440 445

Asn Gly Leu Ser Gly Lys Cys Leu Gln Glu Ala Gln Lys Glu Gly Asn

450 455 460

Ser Ile Leu Pro Glu Arg Arg Gly Arg Pro Glu Ile Ser Leu Asp Glu

465 470 475 480

Arg Gly Glu Gly Gly His Val His Thr Ser Asp Asp Ser Glu Val Val

485 490 495

Phe Ser Ser Cys Asp Leu Asn Leu Thr Met Glu Asp Ser Asp Gly Val

500 505 510

Thr Tyr Ala Leu Lys Cys Asp Ser Ser Gly His Ala Pro Glu Ile Val

515 520 525

Ser Thr Val His Glu Asp Tyr Ser Gly Ser Ser Glu Ser Ser Asn Asp

530 535 540

Glu Ser Asp Ser Glu Asp Thr Asp Ser Asp Asp Ser Ser Ile Pro Arg

545 550 555 560

Asn Arg Leu Gln Ser Val Val Val Val Pro Lys Asn Ser Thr Leu Pro

565 570 575

Met Glu Glu Thr Ser Pro Cys Ser Ser Arg Ser Ser Gln Ser Tyr Arg

580 585 590

His Tyr Ser Asp His Trp Glu Asp Glu Arg Leu Glu Ser Arg Arg His

595 600 605

Leu Tyr Glu Glu Lys Phe Glu Ser Ile Ala Ser Lys Ala Cys Pro Gln

610 615 620

Thr Asp Lys Phe Phe Leu His Lys Gly Thr Glu Lys Asn Pro Glu Ile

625 630 635 640

Ser Phe Thr Gln Ser Ser Arg Lys Gln Ile Asp Asn Arg Leu Pro Glu

645 650 655

Leu Ser His Pro Gln Ser Asp Gly Val Asp Ser Thr Ser His Thr Asp

660 665 670

Val Lys Ser Asp Pro Leu Gly His Pro Asn Ser Glu Glu Thr Val Lys

675 680 685

Ala Lys Ile Pro Ser Arg Gln Gln Glu Glu Leu Pro Ile Tyr Ser Ser

690 695 700

Asp Phe Glu Asp Val Pro Asn Lys Ser Trp Gln Gln Thr Thr Phe Gln

705 710 715 720

Asn Arg Pro Asp Ser Arg Leu Gly Lys Thr Glu Leu Ser Phe Ser Ser

725 730 735

Ser Cys Glu Ile Pro His Val Asp Gly Leu His Ser Ser Glu Glu Leu

740 745 750

Arg Asn Leu Gly Trp Asp Phe Ser Gln Glu Lys Pro Ser Ala Thr Tyr

755 760 765

Gln Gln Pro Asp Ser Ser Tyr Gly Ala Cys Gly Gly His Lys Tyr Gln

770 775 780

Gln Asn Ala Glu Gln Tyr Gly Gly Thr Arg Asp Tyr Trp Gln Gly Asn

785 790 795 800

Gly Tyr Trp Asp Pro Arg Ser Gly Arg Pro Pro Gly Thr Gly Val Val

805 810 815

Tyr Asp Arg Thr Gln Gly Gln Val Pro Asp Ser Leu Thr Asp Asp Arg

820 825 830

Glu Glu Glu Glu Asn Trp Asp Gln Gln Asp Gly Ser His Phe Ser Asp

835 840 845

Gln Ser Asp Lys Phe Leu Leu Ser Leu Gln Lys Asp Lys Gly Ser Val

850 855 860

Gln Ala Pro Glu Ile Ser Ser Asn Ser Ile Lys Asp Thr Leu Ala Val

865 870 875 880

Asn Glu Lys Lys Asp Phe Ser Lys Asn Leu Glu Lys Asn Asp Ile Lys

885 890 895

Asp Arg Gly Pro Leu Lys Lys Arg Arg Gln Glu Ile Glu Ser Asp Ser

900 905 910

Glu Ser Asp Gly Glu Leu Gln Asp Arg Lys Lys Val Arg Val Glu Val

915 920 925

Glu Gln Gly Glu Thr Ser Val Pro Pro Gly Ser Ala Leu Val Gly Pro

930 935 940

Ser Cys Val Met Asp Asp Phe Arg Asp Pro Gln Arg Trp Lys Glu Cys

945 950 955 960

Ala Lys Gln Gly Lys Met Pro Cys Tyr Phe Asp Leu Ile Glu Glu Asn

965 970 975

Val Tyr Leu Thr Glu Arg Lys Lys Asn Lys Ser His Arg Asp Ile Lys

980 985 990

Arg Met Gln Cys Glu Cys Thr Pro Leu Ser Lys Asp Glu Arg Ala Gln

995 1000 1005

Gly Glu Ile Ala Cys Gly Glu Asp Cys Leu Asn Arg Leu Leu Met

1010 1015 1020

Ile Glu Cys Ser Ser Arg Cys Pro Asn Gly Asp Tyr Cys Ser Asn

1025 1030 1035

Arg Arg Phe Gln Arg Lys Gln His Ala Asp Val Glu Val Ile Leu

1040 1045 1050

Thr Glu Lys Lys Gly Trp Gly Leu Arg Ala Ala Lys Asp Leu Pro

1055 1060 1065

Ser Asn Thr Phe Val Leu Glu Tyr Cys Gly Glu Val Leu Asp His

1070 1075 1080

Lys Glu Phe Lys Ala Arg Val Lys Glu Tyr Ala Arg Asn Lys Asn

1085 1090 1095

Ile His Tyr Tyr Phe Met Ala Leu Lys Asn Asp Glu Ile Ile Asp

1100 1105 1110

Ala Thr Gln Lys Gly Asn Cys Ser Arg Phe Met Asn His Ser Cys

1115 1120 1125

Glu Pro Asn Cys Glu Thr Gln Lys Trp Thr Val Asn Gly Gln Leu

1130 1135 1140

Arg Val Gly Phe Phe Thr Thr Lys Leu Val Pro Ser Gly Ser Glu

1145 1150 1155

Leu Thr Phe Asp Tyr Gln Phe Gln Arg Tyr Gly Lys Glu Ala Gln

1160 1165 1170

Lys Cys Phe Cys Gly Ser Ala Asn Cys Arg Gly Tyr Leu Gly Gly

1175 1180 1185

Glu Asn Arg Val Ser Ile Arg Ala Ala Gly Gly Lys Met Lys Lys

1190 1195 1200

Glu Arg Ser Arg Lys Lys Asp Ser

1205 1210

<210>3

<211>1617

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>3

atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60

ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120

tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180

ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240

atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300

gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360

gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420

acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480

gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540

aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600

tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660

ctgatcgaag gtcgtgggat ccccgaattc ccggggatcc ccatggcccg ggcgacgtcg 720

actgatggtg agcttcagga cagaaagaaa gttagagtgg aggtagagca gggagagaca 780

tcagtgcccc caggttcagc actggttggg ccctcctgtg tcatggatga cttcagggac 840

ccacagcgat ggaaggaatg tgccaagcaa gggaaaatgc catgttactt tgatcttatt 900

gaagaaaatg tttatttaac agaaagaaag aagaataaat ctcatcgaga tattaagcga 960

atgcagtgtg agtgtacacc tctttctaaa gatgaaagag ctcaaggtga aatagcatgt 1020

ggggaagatt gtcttaatcg tcttctcatg attgaatgtt cttctcggtg tccaaatggg 1080

gattattgtt ccaatagacg gtttcagaga aaacagcatg cagatgtgga agtcatactc 1140

acagaaaaga aaggctgggg cttgagagct gccaaagacc ttccttcgaa cacctttgtc 1200

ctagaatatt gtggagaggt actcgatcat aaagagttta aagctcgagt gaaggagtat 1260

gcacgaaaca aaaacatcca ttactatttc atggccctga agaatgatga gataatagat 1320

gccactcaaa aaggaaattg ctctcgtttc atgaatcaca gctgtgaacc aaattgtgaa 1380

acccaaaaat ggactgtgaa cggacaactg agggttgggt tttttaccac caaactggtt 1440

ccttcaggct cagagttaac gtttgactat cagttccaga gatatggaaa agaagcccag 1500

aaatgtttct gcggatcagc caattgccgg ggttacctgg gaggagaaaa cagagtcagc 1560

attagagcag caggagggaa aatgaagaag gaacgatctc gtaagaagga ttcatag 1617

<210>4

<211>538

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>4

Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro

1 5 10 15

Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu

20 25 30

Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu

35 40 45

Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys

50 55 60

Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn

65 70 75 80

Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu

85 90 95

Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser

100 105 110

Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu

115 120 125

Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn

130 135 140

Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp

145 150 155 160

Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu

165 170 175

Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr

180 185 190

Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala

195 200 205

Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Ile Glu Gly

210 215 220

Arg Gly Ile Pro Glu Phe Pro Gly Ile Pro Met Ala Arg Ala Thr Ser

225 230 235 240

Thr Asp Gly Glu Leu Gln Asp Arg Lys Lys Val Arg Val Glu Val Glu

245 250 255

Gln Gly Glu Thr Ser Val Pro Pro Gly Ser Ala Leu Val Gly Pro Ser

260 265 270

Cys Val Met Asp Asp Phe Arg Asp Pro Gln Arg Trp Lys Glu Cys Ala

275 280 285

Lys Gln Gly Lys Met Pro Cys Tyr Phe Asp Leu Ile Glu Glu Asn Val

290 295 300

Tyr Leu Thr Glu Arg Lys Lys Asn Lys Ser His Arg Asp Ile Lys Arg

305 310 315 320

Met Gln Cys Glu Cys Thr Pro Leu Ser Lys Asp Glu Arg Ala Gln Gly

325 330 335

Glu Ile Ala Cys Gly Glu Asp Cys Leu Asn Arg Leu Leu Met Ile Glu

340 345 350

Cys Ser Ser Arg Cys Pro Asn Gly Asp Tyr Cys Ser Asn Arg Arg Phe

355 360 365

Gln Arg Lys Gln His Ala Asp Val Glu Val Ile Leu Thr Glu Lys Lys

370 375 380

Gly Trp Gly Leu Arg Ala Ala Lys Asp Leu Pro Ser Asn Thr Phe Val

385 390 395 400

Leu Glu Tyr Cys Gly Glu Val Leu Asp His Lys Glu Phe Lys Ala Arg

405 410 415

Val Lys Glu Tyr Ala Arg Asn Lys Asn Ile His Tyr Tyr Phe Met Ala

420 425 430

Leu Lys Asn Asp Glu Ile Ile Asp Ala Thr Gln Lys Gly Asn Cys Ser

435 440 445

Arg Phe Met Asn His Ser Cys Glu Pro Asn Cys Glu Thr Gln Lys Trp

450 455 460

Thr Val Asn Gly Gln Leu Arg Val Gly Phe Phe Thr Thr Lys Leu Val

465 470 475 480

Pro Ser Gly Ser Glu Leu Thr Phe Asp Tyr Gln Phe Gln Arg Tyr Gly

485 490 495

Lys Glu Ala Gln Lys Cys Phe Cys Gly Ser Ala Asn Cys Arg Gly Tyr

500 505 510

Leu Gly Gly Glu Asn Arg Val Ser Ile Arg Ala Ala Gly Gly Lys Met

515 520 525

Lys Lys Glu Arg Ser Arg Lys Lys Asp Ser

530 535

<210>5

<211>6652

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>5

acagggacct aaggacatca tcctattcta aatctgatcg ggactgtaaa actgagacct 60

cttacttaga gatggaaaga agaggcaagt attcttcaaa actagaaaga gaatctaaaa 120

ggacttcaga aaatgaagca attaaaagat gttgttctcc ccctaatgaa ctgggattcc 180

gacgagggtc atcatattct aagcatgaca gtagtgcttc ccgttataaa tctacccttt 240

caaaacctat acccaagtct gataaattta aaaattcttt ctgttgtaca gaattaaatg 300

aagaaatcaa acaatctcat tcttttagtt tacagacacc ttgttcaaaa ggtagtgaat 360

taagaatgat taataaaaat cctgaaagag aaaaggctgg gtctccagct ccatcaaatc 420

gattaaatga ttcacctact ttaaaaaagc tagatgaatt gcctattttt aagtccgaat 480

ttataacaca tgatagccat gatagtatta aggaattaga ctctttatct aaagtgaaga 540

atgatcaatt aagaagtttt tgtcccatag aattaaatat aaatggatct cctggggcag 600

aatctgattt ggcaacattt tgcacttcta aaactgatgc tgttttaatg acttctgatg 660

atagtgtgac tggatcggaa ttatcccctt tggtcaaagc atgcatgctt tcatcaaatg 720

gatttcagaa tattagtagg tgcaaagaaa aagacttgga tgatacctgc atgctgcata 780

agaagtcaga aagcccattt agagaaacag aacctctggt gtcaccacac caagataaac 840

tcatgtctat gccagttatg actgtggatt attccaaaac agtagttaaa gaaccagttg 900

atacgagggt ttcttgctgc aaaaccaaag attcagacat atactgtact ttgaacgata 960

gcaacccttc tttgtgtaac tctgaagctg aaaatattga gccttcagtt atgaagattt 1020

cttcaaatag ctttatgaat gtgcatttgg aatcaaaacc agttatatgt gatagtagaa 1080

atttgacaga tcactcaaaa tttgcatgtg aagaatataa gcagagcatc ggtagcacta 1140

gttcagcttc tgttaatcat tttgatgatt tatatcaacc tattgggagt tcaggtattg 1200

cttcatctct tcagagtctt ccaccaggaa taaaggtgga cagtctaact ctcttgaaat 1260

gcggagagaa cacatctcca gttctggatg cagtgctaaa gagtaaaaaa agttcagagt 1320

ttttaaagca tgcagggaaa gaaacaatag tagaagtagg tagtgacctt cctgattcag 1380

gaaagggatt tgcttccagg gagaacaggc gtaataatgg gttatctggg aaatgtttgc 1440

aagaggctca aaaagaaggg aattccatat tgcctgaaag aagaggaaga ccagaaatct 1500

ctttagatga aagaggagaa ggaggacatg tgcatacttc tgatgactca gaagttgtat 1560

tttcttcttg tgatttgaat ttaaccatgg aagacagtga tggtgtaact tatgcattaa 1620

agtgtgacag tagtggtcat gccccagaaa ttgtgtctac agttcatgaa gattattctg 1680

gctcttctga aagttcaaat gatgaaagtg attcagaaga tacggattcg gatgatagca 1740

gtattccaag aaaccgtctc cagtctgttg tggttgtgcc aaagaattct actttgccca 1800

tggaagaaac aagtccttgt tcttctcgga gcagtcaaag ttatagacac tattctgacc 1860

attgggaaga tgagagattg gagtcaagga gacatttgta tgaggaaaaa tttgaaagta 1920

tagcaagtaa agcctgtcct caaactgata agtttttcct tcataaagga acagagaaga 1980

atccggaaat ttcttttaca cagtccagta gaaaacaaat agataaccgc ctgcctgaac 2040

tttctcatcc tcagagtgat ggggttgata gtacaagtca tacagatgtg aaatctgacc 2100

ctctgggtca cccaaattca gaggaaaccg tgaaagccaa aataccttct aggcagcaag 2160

aagagctgcc aatttattct tctgattttg aagatgtccc aaataagtct tggcaacaga 2220

ccactttcca aaacaggcca gatagtagac tgggaaaaac agaattgagt ttttcttcct 2280

cttgtgagat accacatgtg gatggcttgc actcatcaga agagctcaga aacttaggtt 2340

gggacttctc tcaagaaaag ccttctgcca cgtatcagca acctgacagt agctatggag 2400

cttgtggtgg acacaagtat cagcaaaatg cagaacagta tggtgggaca cgtgattact 2460

ggcaaggcaa tggttactgg gatccaagat caggtagacc tcctggaact ggggttgtgt 2520

atgatcgaac tcaaggacaa gtaccagatt ccctaacaga tgatcgtgaa gaagaggaga 2580

attgggatca acaggatgga tcccattttt cagaccagtc cgataaattt cttctatccc 2640

ttcagaaaga caaggggtca gtgcaagcac ctgaaataag cagcaattcc attaaggaca 2700

ctttagctgt gaatgaaaag aaagattttt caaaaaactt agaaaaaaat gatatcaaag 2760

atagagggcc tcttaaaaaa aggaggcagg aaatagagag tgattctgaa agtgatggtg 2820

agcttcagga cagaaagaaa gttagagtgg aggtagagca gggagagaca tcagtgcccc 2880

caggttcagc actggttggg ccctcctgtg tcatggatga cttcagggac ccacagcgat 2940

ggaaggaatg tgccaagcaa gggaaaatgc catgttactt tgatcttatt gaagaaaatg 3000

tttatttaac agaaagaaag aagaataaat ctcatcgaga tattaagcga atgcagtgtg 3060

agtgtacacc tctttctaaa gatgaaagag ctcaaggtga aatagcatgt ggggaagatt 3120

gtcttaatcg tcttctcatg attgaatgtt cttctcggtg tccaaatggg gattattgtt 3180

ccaatagacg gtttcagaga aaacagcatg cagatgtgga agtcatactc acagaaaaga 3240

aaggctgggg cttgagagct gccaaagacc ttccttcgaa cacctttgtc ctagaatatt 3300

gtggagaggt actcgatcat aaagagttta aagctcgagt gaaggagtat gcacgaaaca 3360

aaaacatcca ttactatttc atggccctga agaatgatga gataatagat gccactcaaa 3420

aaggaaattg ctctcgtttc atgaatcaca gctgtgaacc aaattgtgaa acccaaaaat 3480

ggactgtgaa cggacaactg agggttgggt tttttaccac caaactggtt ccttcaggct 3540

cagagttaac gtttgactat cagttccaga gatatggaaa agaagcccag aaatgtttct 3600

gcggatcagc caattgccgg ggttacctgg gaggagaaaa cagagtcagc attagagcag 3660

caggagggaa aatgaagaag gaacgatctc gtaagaagga ttcagtggat ggagagctag 3720

aagctctgat ggaaaatggt gagggtctct ctgataaaaa ccaggtgccc agcttatccc 3780

ggctaatggt tagaattgaa actttggagc agaaacttac ctgtctggaa ctcatacaga 3840

acacacactc acagtcctgc ctgaagtcct ttctggaacg tcatgggctg tctttgttgt 3900

ggatctggat ggcagagcta ggtgacggcc gggaaagtaa ccagaagctt caggaagaga 3960

ttataaagac tttggaacac ttgcccattc ctactaaaaa tatgttggag gaaagcaaag 4020

tacttccaat tattcaacgc tggtctcaga ctaagactgc tgtccctccg ttgagtgaag 4080

gagatgggta ttctagtgag aatacatcgc gtgctcatac accactcaac acacctgatc 4140

cttccaccaa gctgagcaca gaagctgaca cagacactcc caagaaacta atgtttcgca 4200

gactgaaaat tataagtgaa aatagcatgg acagtgcaat ctctgatgca accagtgagc 4260

tagaaggcaa ggatggcaaa gaggatcttg atcaattaga aaatgtccct gtagaggaag 4320

aggaagaatt gcagtcacaa cagctactcc cacaacagct gcctgaatgc aaagttgata 4380

gtgaaaccaa catagaagct agtaagctac ctacatctga accagaagct gacgctgaaa 4440

tagagcccaa agagagcaac ggcacaaaac tagaagaacc tattaatgaa gaaacaccat 4500

cccaagatga agaggagggt gtgtctgatg tggagagtga aaggagccaa gaacagccag 4560

ataaaacagt ggatataagt gatttggcca ccaaactcct ggacagttgg aaagacctaa 4620

aggaggtata tcgaattcca aagaaaagtc aaactgaaaa ggaaaacaca acaactgaac 4680

gaggaaggga tgctgttggc ttcagagatc aaacacctgc cccgaagact cctaataggt 4740

caagagagag agacccagac aagcaaactc aaaataaaga gaaaaggaaa cgaagaagct 4800

ccctctcacc accctcttct gcctatgagc ggggaacaaa aaggccagat gacagatatg 4860

atacaccaac ttctaaaaag aaagtacgaa ttaaagaccg caataaactt tctacagagg 4920

aacgccggaa gttgtttgag caagaggtgg ctcaacggga ggctcagaaa caacagcaac 4980

agatgcagaa cctgggaatg acatcaccac tgccctatga ctctcttggt tataatgccc 5040

cgcatcatcc ctttgctggt tacccaccag gttatcccat gcaggcctat gtggatccca 5100

gcaaccctaa tgctggaaag gtgctcctgc ccacacccag catggaccca gtgtgttctc 5160

ctgctcctta tgatcatgct cagcccttgg tgggacattc tacagaaccc ctttctgccc 5220

ctccaccagt accagtggtg ccacatgtgg cagctcctgt ggaagtttcc agttcccagt 5280

atgtggccca gagtgatggt gtagtacacc aagactccag cgttgctgtc ttgccagtgc 5340

cggcccccgg cccagttcag ggacagaatt atagtgtttg ggattcaaac caacagtctg 5400

tcagtgtaca gcagcagtac tctcctgcac agtctcaagc aaccatatat tatcaaggac 5460

agacatgtcc aacagtctat ggtgtgacat caccttattc acagacaact ccaccaattg 5520

tacagagtta tgcccagcca agtcttcagt atatccaggg gcaacagatt ttcacagctc 5580

atccacaagg agtggtggta cagccagccg cagcagtgac tacaatagtt gcaccagggc 5640

agcctcagcc cttgcagcca tctgaaatgg ttgtgacaaa taatctcttg gatctgccgc 5700

ccccctctcc tcccaaacca aaaaccattg tcttacctcc caactggaag acagctcgag 5760

atccagaagg gaagatttat tactaccatg tgatcacaag gcagactcag tgggatcctc 5820

ctacttggga aagcccagga gatgatgcca gccttgagca tgaagctgag atggacctgg 5880

gaactccaac atatgatgaa aaccccatga aggcctcgaa aaagcccaag acagcagaag 5940

cagacacctc cagtgaacta gcaaagaaaa gcaaagaagt attcagaaaa gagatgtccc 6000

agttcatcgt ccagtgcctg aacccttacc ggaaacctga ctgcaaagtg ggaagaatta 6060

ccacaactga agactttaaa catctggctc gcaagctgac tcacggtgtt atgaataagg 6120

agctgaagta ctgtaagaat cctgaggacc tggagtgcaa tgagaatgtg aaacacaaaa 6180

ccaaggagta cattaagaag tacatgcaga agtttggggc tgtttacaaa cccaaagagg 6240

acactgaatt agagtgactg ttgggccagg gtgggaggat gggtggtcag gtaagacaga 6300

ctctagggag aggaaatcct gtgggccttt ctgtcccacc cctgtcagca ctgtgctact 6360

gatgatacat caccctgggg aattcaaccc tgcagatgtc aactgaaggc cacaaaaatg 6420

aactccatct acaagtgatt acctagttgt gagctgttgg catgtggtta gaagccatca 6480

gaggtgcaag ggcttagaaa agaccctggc cagacctgac tccactctta aacctgggtc 6540

ttctccttgg cggtgctgtc agcgcacaga cccatgcgca tccccaccca caacccttta 6600

ccctgatgat ctgtattata ttttaatgta tatgtgaata tattgaaaat aa 6652

<210>6

<211>2061

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>6

Met Glu Arg Arg Gly Lys Tyr Ser Ser Lys Leu Glu Arg Glu Ser Lys

1 5 10 15

Arg Thr Ser Glu Asn Glu Ala Ile Lys Arg Cys Cys Ser Pro Pro Asn

20 25 30

Glu Leu Gly Phe Arg Arg Gly Ser Ser Tyr Ser Lys His Asp Ser Ser

35 40 45

Ala Ser Arg Tyr Lys Ser Thr Leu Ser Lys Pro Ile Pro Lys Ser Asp

50 55 60

Lys Phe Lys Asn Ser Phe Cys Cys Thr Glu Leu Asn Glu Glu Ile Lys

65 70 75 80

Gln Ser His Ser Phe Ser Leu Gln Thr Pro Cys Ser Lys Gly Ser Glu

85 90 95

Leu Arg Met Ile Asn Lys Asn Pro Glu Arg Glu Lys Ala Gly Ser Pro

100 105 110

Ala Pro Ser Asn Arg Leu Asn Asp Ser Pro Thr Leu Lys Lys Leu Asp

115 120 125

Glu Leu Pro Ile Phe Lys Ser Glu Phe Ile Thr His Asp Ser His Asp

130 135 140

Ser Ile Lys Glu Leu Asp Ser Leu Ser Lys Val Lys Asn Asp Gln Leu

145 150 155 160

Arg Ser Phe Cys Pro Ile Glu Leu Asn Ile Asn Gly Ser Pro Gly Ala

165 170 175

Glu Ser Asp Leu Ala Thr Phe Cys Thr Ser Lys Thr Asp Ala Val Leu

180 185 190

Met Thr Ser Asp Asp Ser Val Thr Gly Ser Glu Leu Ser Pro Leu Val

195 200 205

Lys Ala Cys Met Leu Ser Ser Asn Gly Phe Gln Asn Ile Ser Arg Cys

210 215 220

Lys Glu Lys Asp Leu Asp Asp Thr Cys Met Leu His Lys Lys Ser Glu

225 230 235 240

Ser Pro Phe Arg Glu Thr Glu Pro Leu Val Ser Pro His Gln Asp Lys

245 250 255

Leu Met Ser Met Pro Val Met Thr Val Asp Tyr Ser Lys Thr Val Val

260 265 270

Lys Glu Pro Val Asp Thr Arg Val Ser Cys Cys Lys Thr Lys Asp Ser

275 280 285

Asp Ile Tyr Cys Thr Leu Asn Asp Ser Asn Pro Ser Leu Cys Asn Ser

290 295 300

Glu Ala Glu Asn Ile Glu Pro Ser Val Met Lys Ile Ser Ser Asn Ser

305 310 315 320

Phe Met Asn Val His Leu Glu Ser Lys Pro Val Ile Cys Asp Ser Arg

325 330 335

Asn Leu Thr Asp His Ser Lys Phe Ala Cys Glu Glu Tyr Lys Gln Ser

340 345 350

Ile Gly Ser Thr Ser Ser Ala Ser Val Asn His Phe Asp Asp Leu Tyr

355 360 365

Gln Pro Ile Gly Ser Ser Gly Ile Ala Ser Ser Leu Gln Ser Leu Pro

370 375 380

Pro Gly Ile Lys Val Asp Ser Leu Thr Leu Leu Lys Cys Gly Glu Asn

385 390 395 400

Thr Ser Pro Val Leu Asp Ala Val Leu Lys Ser Lys Lys Ser Ser Glu

405 410 415

Phe Leu Lys His Ala Gly Lys Glu Thr Ile Val Glu Val Gly Ser Asp

420 425 430

Leu Pro Asp Ser Gly Lys Gly Phe Ala Ser Arg Glu Asn Arg Arg Asn

435 440 445

Asn Gly Leu Ser Gly Lys Cys Leu Gln Glu Ala Gln Lys Glu Gly Asn

450 455 460

Ser Ile Leu Pro Glu Arg Arg Gly Arg Pro Glu Ile Ser Leu Asp Glu

465 470 475 480

Arg Gly Glu Gly Gly His Val His Thr Ser Asp Asp Ser Glu Val Val

485 490 495

Phe Ser Ser Cys Asp Leu Asn Leu Thr Met Glu Asp Ser Asp Gly Val

500 505 510

Thr Tyr Ala Leu Lys Cys Asp Ser Ser Gly His Ala Pro Glu Ile Val

515 520 525

Ser Thr Val His Glu Asp Tyr Ser Gly Ser Ser Glu Ser Ser Asn Asp

530 535 540

Glu Ser Asp Ser Glu Asp Thr Asp Ser Asp Asp Ser Ser Ile Pro Arg

545 550 555 560

Asn Arg Leu Gln Ser Val Val Val Val Pro Lys Asn Ser Thr Leu Pro

565 570 575

Met Glu Glu Thr Ser Pro Cys Ser Ser Arg Ser Ser Gln Ser Tyr Arg

580 585 590

His Tyr Ser Asp His Trp Glu Asp Glu Arg Leu Glu Ser Arg Arg His

595 600 605

Leu Tyr Glu Glu Lys Phe Glu Ser Ile Ala Ser Lys Ala Cys Pro Gln

610 615 620

Thr Asp Lys Phe Phe Leu His Lys Gly Thr Glu Lys Asn Pro Glu Ile

625 630 635 640

Ser Phe Thr Gln Ser Ser Arg Lys Gln Ile Asp Asn Arg Leu Pro Glu

645 650 655

Leu Ser His Pro Gln Ser Asp Gly Val Asp Ser Thr Ser His Thr Asp

660 665 670

Val Lys Ser Asp Pro Leu Gly His Pro Asn Ser Glu Glu Thr Val Lys

675 680 685

Ala Lys Ile Pro Ser Arg Gln Gln Glu Glu Leu Pro Ile Tyr Ser Ser

690 695 700

Asp Phe Glu Asp Val Pro Asn Lys Ser Trp Gln Gln Thr Thr Phe Gln

705 710 715 720

Asn Arg Pro Asp Ser Arg Leu Gly Lys Thr Glu Leu Ser Phe Ser Ser

725 730 735

Ser Cys Glu Ile Pro His Val Asp Gly Leu His Ser Ser Glu Glu Leu

740 745 750

Arg Asn Leu Gly Trp Asp Phe Ser Gln Glu Lys Pro Ser Ala Thr Tyr

755 760 765

Gln Gln Pro Asp Ser Ser Tyr Gly Ala Cys Gly Gly His Lys Tyr Gln

770 775 780

Gln Asn Ala Glu Gln Tyr Gly Gly Thr Arg Asp Tyr Trp Gln Gly Asn

785 790 795 800

Gly Tyr Trp Asp Pro Arg Ser Gly Arg Pro Pro Gly Thr Gly Val Val

805 810 815

Tyr Asp Arg Thr Gln Gly Gln Val Pro Asp Ser Leu Thr Asp Asp Arg

820 825 830

Glu Glu Glu Glu Asn Trp Asp Gln Gln Asp Gly Ser His Phe Ser Asp

835 840 845

Gln Ser Asp Lys Phe Leu Leu Ser Leu Gln Lys Asp Lys Gly Ser Val

850 855 860

Gln Ala Pro Glu Ile Ser Ser Asn Ser Ile Lys Asp Thr Leu Ala Val

865 870 875 880

Asn Glu Lys Lys Asp Phe Ser Lys Asn Leu Glu Lys Asn Asp Ile Lys

885 890 895

Asp Arg Gly Pro Leu Lys Lys Arg Arg Gln Glu Ile Glu Ser Asp Ser

900 905 910

Glu Ser Asp Gly Glu Leu Gln Asp Arg Lys Lys Val Arg Val Glu Val

915 920 925

Glu Gln Gly Glu Thr Ser Val Pro Pro Gly Ser Ala Leu Val Gly Pro

930 935 940

Ser Cys Val Met Asp Asp Phe Arg Asp Pro Gln Arg Trp Lys Glu Cys

945 950 955 960

Ala Lys Gln Gly Lys Met Pro Cys Tyr Phe Asp Leu Ile Glu Glu Asn

965 970 975

Val Tyr Leu Thr Glu Arg Lys Lys Asn Lys Ser His Arg Asp Ile Lys

980 985 990

Arg Met Gln Cys Glu Cys Thr Pro Leu Ser Lys Asp Glu Arg Ala Gln

995 1000 1005

Gly Glu Ile Ala Cys Gly Glu Asp Cys Leu Asn Arg Leu Leu Met

1010 1015 1020

Ile Glu Cys Ser Ser Arg Cys Pro Asn Gly Asp Tyr Cys Ser Asn

1025 1030 1035

Arg Arg Phe Gln Arg Lys Gln His Ala Asp Val Glu Val Ile Leu

1040 1045 1050

Thr Glu Lys Lys Gly Trp Gly Leu Arg Ala Ala Lys Asp Leu Pro

1055 1060 1065

Ser Asn Thr Phe Val Leu Glu Tyr Cys Gly Glu Val Leu Asp His

1070 1075 1080

Lys Glu Phe Lys Ala Arg Val Lys Glu Tyr Ala Arg Asn Lys Asn

1085 1090 1095

Ile His Tyr Tyr Phe Met Ala Leu Lys Asn Asp Glu Ile Ile Asp

1100 1105 1110

Ala Thr Gln Lys Gly Asn Cys Ser Arg Phe Met Asn His Ser Cys

1115 1120 1125

Glu Pro Asn Cys Glu Thr Gln Lys Trp Thr Val Asn Gly Gln Leu

1130 1135 1140

Arg Val Gly Phe Phe Thr Thr Lys Leu Val Pro Ser Gly Ser Glu

1145 1150 1155

Leu Thr Phe Asp Tyr Gln Phe Gln Arg Tyr Gly Lys Glu Ala Gln

1160 1165 1170

Lys Cys Phe Cys Gly Ser Ala Asn Cys Arg Gly Tyr Leu Gly Gly

1175 1180 1185

Glu Asn Arg Val Ser Ile Arg Ala Ala Gly Gly Lys Met Lys Lys

1190 1195 1200

Glu Arg Ser Arg Lys Lys Asp Ser Val Asp Gly Glu Leu Glu Ala

1205 1210 1215

Leu Met Glu Asn Gly Glu Gly Leu Ser Asp Lys Asn Gln Val Pro

1220 1225 1230

Ser Leu Ser Arg Leu Met Val Arg Ile Glu Thr Leu Glu Gln Lys

1235 1240 1245

Leu Thr Cys Leu Glu Leu Ile Gln Asn Thr His Ser Gln Ser Cys

1250 1255 1260

Leu Lys Ser Phe Leu Glu Arg His Gly Leu Ser Leu Leu Trp Ile

1265 1270 1275

Trp Met Ala Glu Leu Gly Asp Gly Arg Glu Ser Asn Gln Lys Leu

1280 1285 1290

Gln Glu Glu Ile Ile Lys Thr Leu Glu His Leu Pro Ile Pro Thr

1295 1300 1305

Lys Asn Met Leu Glu Glu Ser Lys Val Leu Pro Ile Ile Gln Arg

1310 1315 1320

Trp Ser Gln Thr Lys Thr Ala Val Pro Pro Leu Ser Glu Gly Asp

1325 1330 1335

Gly Tyr Ser Ser Glu Asn Thr Ser Arg Ala His Thr Pro Leu Asn

1340 1345 1350

Thr Pro Asp Pro Ser Thr Lys Leu Ser Thr Glu Ala Asp Thr Asp

1355 1360 1365

Thr Pro Lys Lys Leu Met Phe Arg Arg Leu Lys Ile Ile Ser Glu

1370 1375 1380

Asn Ser Met Asp Ser Ala Ile Ser Asp Ala Thr Ser Glu Leu Glu

1385 1390 1395

Gly Lys Asp Gly Lys Glu Asp Leu Asp Gln Leu Glu Asn Val Pro

1400 1405 1410

Val Glu Glu Glu Glu Glu Leu Gln Ser Gln Gln Leu Leu Pro Gln

1415 1420 1425

Gln Leu Pro Glu Cys Lys Val Asp Ser Glu Thr Asn Ile Glu Ala

1430 1435 1440

Ser Lys Leu Pro Thr Ser Glu Pro Glu Ala Asp Ala Glu Ile Glu

1445 1450 1455

Pro Lys Glu Ser Asn Gly Thr Lys Leu Glu Glu Pro Ile Asn Glu

1460 1465 1470

Glu Thr Pro Ser Gln Asp Glu Glu Glu Gly Val Ser Asp Val Glu

1475 1480 1485

Ser Glu Arg Ser Gln Glu Gln Pro Asp Lys Thr Val Asp Ile Ser

1490 1495 1500

Asp Leu Ala Thr Lys Leu Leu Asp Ser Trp Lys Asp Leu Lys Glu

1505 1510 1515

Val Tyr Arg Ile Pro Lys Lys Ser Gln Thr Glu Lys Glu Asn Thr

1520 1525 1530

Thr Thr Glu Arg Gly Arg Asp Ala Val Gly Phe Arg Asp Gln Thr

1535 1540 1545

Pro Ala Pro Lys Thr Pro Asn Arg Ser Arg Glu Arg Asp Pro Asp

1550 1555 1560

Lys Gln Thr Gln Asn Lys Glu Lys Arg Lys Arg Arg Ser Ser Leu

1565 1570 1575

Ser Pro Pro Ser Ser Ala Tyr Glu Arg Gly Thr Lys Arg Pro Asp

1580 1585 1590

Asp Arg Tyr Asp Thr Pro Thr Ser Lys Lys Lys Val Arg Ile Lys

1595 1600 1605

Asp Arg Asn Lys Leu Ser Thr Glu Glu Arg Arg Lys Leu Phe Glu

1610 1615 1620

Gln Glu Val Ala Gln Arg Glu Ala Gln Lys Gln Gln Gln Gln Met

1625 1630 1635

Gln Asn Leu Gly Met Thr Ser Pro Leu Pro Tyr Asp Ser Leu Gly

1640 1645 1650

Tyr Asn Ala Pro His His Pro Phe Ala Gly Tyr Pro Pro Gly Tyr

1655 1660 1665

Pro Met Gln Ala Tyr Val Asp Pro Ser Asn Pro Asn Ala Gly Lys

1670 1675 1680

Val Leu Leu Pro Thr Pro Ser Met Asp Pro Val Cys Ser Pro Ala

1685 1690 1695

Pro Tyr Asp His Ala Gln Pro Leu Val Gly His Ser Thr Glu Pro

1700 1705 1710

Leu Ser Ala Pro Pro Pro Val Pro Val Val Pro His Val Ala Ala

1715 1720 1725

Pro Val Glu Val Ser Ser Ser Gln Tyr Val Ala Gln Ser Asp Gly

1730 1735 1740

Val Val His Gln Asp Ser Ser Val Ala Val Leu Pro Val Pro Ala

1745 1750 1755

Pro Gly Pro Val Gln Gly Gln Asn Tyr Ser Val Trp Asp Ser Asn

1760 1765 1770

Gln Gln Ser Val Ser Val Gln Gln Gln Tyr Ser Pro Ala Gln Ser

1775 1780 1785

Gln Ala Thr Ile Tyr Tyr Gln Gly Gln Thr Cys Pro Thr Val Tyr

1790 1795 1800

Gly Val Thr Ser Pro Tyr Ser Gln Thr Thr Pro Pro Ile Val Gln

1805 1810 1815

Ser Tyr Ala Gln Pro Ser Leu Gln Tyr Ile Gln Gly Gln Gln Ile

1820 1825 1830

Phe Thr Ala His Pro Gln Gly Val Val Val Gln Pro Ala Ala Ala

1835 1840 1845

Val Thr Thr Ile Val Ala Pro Gly Gln Pro Gln Pro Leu Gln Pro

1850 1855 1860

Ser Glu Met Val Val Thr Asn Asn Leu Leu Asp Leu Pro Pro Pro

1865 1870 1875

Ser Pro Pro Lys Pro Lys Thr Ile Val Leu Pro Pro Asn Trp Lys

1880 1885 1890

Thr Ala Arg Asp Pro Glu Gly Lys Ile Tyr Tyr Tyr His Val Ile

1895 1900 1905

Thr Arg Gln Thr Gln Trp Asp Pro Pro Thr Trp Glu Ser Pro Gly

1910 1915 1920

Asp Asp Ala Ser Leu Glu His Glu Ala Glu Met Asp Leu Gly Thr

1925 1930 1935

Pro Thr Tyr Asp Glu Asn Pro Met Lys Ala Ser Lys Lys Pro Lys

1940 1945 1950

Thr Ala Glu Ala Asp Thr Ser Ser Glu Leu Ala Lys Lys Ser Lys

1955 1960 1965

Glu Val Phe Arg Lys Glu Met Ser Gln Phe Ile Val Gln Cys Leu

1970 1975 1980

Asn Pro Tyr Arg Lys Pro Asp Cys Lys Val Gly Arg Ile Thr Thr

1985 1990 1995

Thr Glu Asp Phe Lys His Leu Ala Arg Lys Leu Thr His Gly Val

2000 2005 2010

Met Asn Lys Glu Leu Lys Tyr Cys Lys Asn Pro Glu Asp Leu Glu

2015 2020 2025

Cys Asn Glu Asn Val Lys His Lys Thr Lys Glu Tyr Ile Lys Lys

2030 2035 2040

Tyr Met Gln Lys Phe Gly Ala Val Tyr Lys Pro Lys Glu Asp Thr

2045 2050 2055

Glu Leu Glu

2060

<210>7

<211>32

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>7

ctcagatcta acagggacct aaggacatca tc 32

<210>8

<211>38

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>8

cgcggtacct tattttcaat atattcacat atacatta 38

<210>9

<211>31

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>9

gcgtcgacgt gatggtgagc ttcaggacag a 31

<210>10

<211>32

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>10

aactgcagat gtgaggcaga caagtcattc ca 32

Claims (10)

1. isolating people HSPC069SET polypeptide is characterized in that this polypeptide is selected from down group:

(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence;

(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have the histone methyl forwarding function by (a) polypeptides derived,

Supplementary condition are that described polypeptide does not have the aminoacid sequence shown in the SEQ ID NO:6.

2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is the polypeptide with SEQ ID NO:2 or 4 aminoacid sequences.

3. isolating polynucleotide is characterized in that, this polynucleotide sequence is selected from down group:

(a) polynucleotide of polypeptide according to claim 1 of encoding;

(b) with polynucleotide (a) complementary polynucleotide.

4. polynucleotide as claimed in claim 3 is characterized in that, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.

5. polynucleotide as claimed in claim 3 is characterized in that, the sequence of these polynucleotide is selected from down a kind of of group:

(a) has the sequence of 72-3704 position among the SEQ ID NO:1;

(b) has the sequence of 1-6731 position among the SEQ ID NO:1;

(c) has the sequence of 1-1614 position among the SEQ ID NO:3.

6. a carrier is characterized in that, it contains the described polynucleotide of claim 3.

7. a genetically engineered host cell is characterized in that, it contains the described carrier of claim 6.

8. the preparation method of a peptide species is characterized in that, this method comprises:

(a) under conditions suitable for the expression, cultivate the described host cell of claim 7;

(b) from culture, isolate people HSPC069SET protein polypeptide.

9. energy and the described people HSPC069SET of claim 1 protein-specific bonded antibody.

10. whether there is the proteic method of HSPC069SET in a test sample, it is characterized in that, may further comprise the steps:

The described antibody of sample and claim 9 is contacted,

Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample HSPC069SET albumen.